(1) Goals of project: - To study the activities of monoclonal antibodies (mAbs) raised against HIV-1 envelope (gp120), CD4, gp120/CD4 complexes, or the HIV-1 co-receptors, on HIV-1 fusion/infection of primary human cells. - To examine the effects of such mAbs on the normal biological functions of primary human cells. - To compare the HIV neutralization capacity of different HIVIG subclasses (2) Experimental approach: - We have collected a panel of murine mAbs specific for the HIV-1 gp 120 or for the CD4 molecules, that bind with increased avidity to the gp120/CD4 complex and may be involved in co-receptor recruitment. - These mAbs were tested for their effects on HIV-1 fusion/infection (both M-tropic and T-tropic strains), and for their activity in various assays designed to measure the formation of tri-molecular complexes between gp120,CD4, and the HIV-1 co-receptors. - HIVIG was fractionated into IGG1, IGG2, and IGG3 subclasses using preparative Protein A column. The purified subclasses were tested in a variety of binding assays ans HIV fusion inhibition and neutralization assays. (3) Major Findings: - CD4-specific mAbs (CG1,7,8) that bind with 10 fold higher avidity to preformed CD4/gp120 complexes than to CD4, were tested in multiple assays of HIV-1 co-receptor (CXCR4 or CCR5) recruitment. In three different assays these mAbs were found to stabilize the association of co-receptor, gp120, and CD4 in tri-molecular comlexes. Such antibodies may be used as tools to disect the steps leading to HIV-1 cell entry. In vivo, they may lead to an increase in viral-cell entry resulting in increased viral loads. - Using HIVIG preparations that were used in human trials, we demonstrated that the IGG3 subclass (which reperesents only 10% of the total IG in the original HIVIG preparation) is superior to other subclasses in its abilty to block HIV-1 mediated fusion and to neutralize multiple cell-free HIV-1 strains, including primary isolates. This superior biological activity may be explained by the unusually long "Hinge" region, unique to IGG3 molecules.